Transcription factor NF-KB activation-dependent ICAM-1 expression in pulmonary endothelial cells results in neutrophil (PMN) adhesion-mediated lung vascular injury. NF-KB-dependent expression of the NF-KB negative regulatory protein A20 inhibits unrestrained activation of NF-KB and thereby controls the inflammatory response. The pre-transcriptional mechanisms involved in regulating A20 gene expression are poorly understood. This research proposal stems from our seminal discovery of multiple "GTCA" sequences (ORE elements) forthe Ca2+ binding transcriptional repressor protein Dream in the 5'-regulatory region of both human and mouse A20 genes. Thus, in Project 3, we will address the crucial role of Dream in regulating the expression of A20 in pulmonary microvessels and thereby controlling NF-KB activation, ICAM-1 expression, and PMN adhesion-mediated lung vascular injury. We cloned a 1.5 kb human A20 promoter sequence linked to reporter and showed robust promoter activity in response to thrombin and TNF-a which was blocked by co-expression of Dream. In addition, we showed that Ca2+ influx through TRPC4 channels or oxidant-sensitive TRPM2 channels induced the de-repression of A20 transcription in response to thrombin and H2O2 respectively. Importantly, we observed that in Dream knock-out (Dream -/-) mice, basal A20 expression was enhanced and this was associated with prevention of thrombin-TNF-a- or LPS-induced ICAM-1 expression in lungs of Dream'''mice. Further, LPS-induced acute lung injury was markedly reduced in Dream -/- mice. In addition, we observed that NF-KB activation in response to inflammatory stimuli was suppressed in Dream-/- mice. Based on these novel findings, in Aim 1, we will test the hypothesis that repressor Dream regulates the expression of the NF-KB inhibitor A20 by interacting with 5'-regulatory region of the A20 gene.
In Aim 2, we will test the hypothesis that H2O2 activation of oxidant-sensitive TRPM2 channels and induces Ca2+ influx leading to de-repression of A20 transcription and its consequent suppression of NF-KB activity.
In Aim 3, we will test the hypothesis that Dream in vivo regulates the basal expression of A20 and thereby suppresses unrestrained activation of NF-KB utilizing Dream-/- mice. With this understanding of Dream, we will be in the position to develop therapeutic strategies that can target Dream function and thereby prevent acute lung injury without compromising innate immunity.
This Project seeks to explore an important signaling pathway that may allow intervention in cases of Acute Lung Injury. We have identified a regulatory protein Dream which on further study may reveal how regulation of Dream may be used to suppress uncontrolled activation of the proinflammatory transcription factor NF-KB in lung.
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